Determination of 4-hydroxyproline in collagen by gas chromatography/mass spectrometry.
Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.
Tredget
E E
EE
Department of Surgery, University of Alberta, Edmonton, Canada.
Falk
N
N
Scott
P G
PG
Hogg
A M
AM
Burke
J F
JF
D000089:Acetic Anhydrides; D002138:Calibration; D003094:Collagen / Q000737:chemistry*; D005463:Fluoroacetates; D008401:Gas Chromatography-Mass Spectrometry; D006909:Hydroxyproline / Q000737:chemistry*; D000432:Methanol
D000089:Acetic Anhydrides; D005463:Fluoroacetates; C017958:trifluoroacetic anhydride; D003094:Collagen; D006909:Hydroxyproline; D000432:Methanol